Methods and compositions for promoting activity of anti-cancer therapies

ABSTRACT

The present invention relates to a method of inhibiting growth of a cancerous cell. The method includes the step of exposing the cancerous cell to an anti-cancer therapy and an effective amount of a steroid saponin.

This application claims priority from Australian Provisional PatentApplication No. 2006904193 filed on 3 Aug. 2006, the contents of whichare to be taken as incorporated herein by this reference.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for inhibitingthe growth of cancerous cells.

BACKGROUND OF THE INVENTION

Chemotherapy and radiation therapy continue to be the main approaches totherapeutic treatment of cancer, with surgery providing the means ofphysically excising the cancer. More recently, biological agents such asantibodies have been developed as anti-cancer therapies.

The application of many anti-cancer agents and radiation therapy hasbeen based on the premise that the cell death caused by the treatmentwith these anti-cancer therapies will bring biological processes intoplay that result in the cancerous cells being ultimately destroyed.

One of these processes is apoptosis. Apoptosis is the complex cellularprogram of self destruction, triggered by a variety of stimuli thatresults in self destruction where dying cells shrink, condense and thenfragment, releasing small membrane-bound apoptotic bodies that arenormally engulfed by other cells such as phagocytes.

Conventional chemotherapeutic agents covalently bond with DNA to formadducts, thereby resulting in DNA damage, and triggering apoptosis.Traditional chemotherapeutic agents suffer from two major disadvantages:(i) they cause severe side effects, because they also affect healthyproliferating cells; and (ii) increased resistance to the agents by thecancerous cells. In this regard, cancer cells have the ability todevelop resistance to the chemotherapeutic agents over time, andultimately may develop multi-drug resistance.

Inhibition of apoptosis in drug resistant tumours not only affects thedeath-inducing activities of the drug, but also allows for thepossibility of cells acquiring additional mutations following DNAdamage. In principle, these mutagenised cells can become more malignantand even less sensitive to subsequent therapies, such that treatment ofhighly resistant tumours containing anti-apoptotic lesions may do moreharm than good.

One of the hallmarks of cancer cells is that they evade apoptosis.Disruption of the apoptotic pathway has important effects on theclinical outcome of chemotherapy. In order for chemotherapeutic agentsto be effective, cells must be capable of undergoing apoptosis.Apoptosis is therefore a vitally important phenomenon in cancerchemotherapy, because many anti-cancer drugs exert their initialantitumour effect against cancer cells by inducing apoptosis.

However, not only can some chemotherapeutic drugs inhibit apoptosisafter a short period of time, but many tumours also have defectiveapoptotic pathways and as such are inherently more resistant tochemotherapy. Furthermore, although the rate of apoptosis is notnecessarily high in tumour tissues, the induction of apoptosis iscorrelated with tumour response and clinical outcome in cancer patients.

One of the major obstacles to treatment of many types of cancer is thedevelopment or presence of resistance to chemotherapeutic agents, suchas occurs in non-small cell lung cancer. For example, the development ofcisplatin resistance is a major cause of treatment failure. Severalmechanisms have been implicated in cisplatin resistance, one of which isaltered expression of oncogenes (e.g. Bcl-2) that subsequently suppressapoptotic pathways and may also contribute to development of resistance.

Accordingly there is a need for agents that may be used in conjunctionwith anti-cancer therapies to enhance their activity against cancerouscells. The present invention relates to the use of steroid saponins topromote the activity of anti-cancer agents and anti-cancer treatments.

A reference herein to a patent document or other matter which is givenas prior art is not to be taken as an admission that the document ormatter was known or that the information it contains was part of thecommon general knowledge as at the priority date of any of the claims.

SUMMARY OF THE INVENTION

The present invention arises out of studies into the ability of steroidsaponins to inhibit the growth of cancerous cells. In particular, it hasbeen found that steroid saponins enhance the activity of a number ofchemotherapeutic and anti-cancer agents to inhibit growth of cancerouscells.

Without being bound by theory, the ability of steroid saponins toenhance the anti-cancer activity of such agents is likely to be due tothe ability of the steroid saponin to promote apoptosis in the cancerouscells when used with the anti-cancer therapy. One mechanism for theability of the steroid saponin to promote apoptosis may be due to theability of the steroid saponin to target or inhibit molecules that mayotherwise suppress apoptosis in cancerous cells.

Thus, the present invention may be used to promote the activity ofanti-cancer agents (such as chemotherapeutic agents) and to promote theactivity of anti-cancer treatments (such as radiotherapy).

Accordingly, the present invention provides a method of inhibitinggrowth of a cancerous cell, the method including exposing the cancerouscell to an anti-cancer therapy and an effective amount of a steroidsaponin.

The present invention also provides use of a steroid saponin and ananti-cancer agent in the preparation of a medicament for inhibitinggrowth of a cancerous cell in a subject.

The present invention also provides a method of promoting the activityof an anti-cancer therapy in a subject, the method including exposingthe subject to an effective amount of a steroid saponin.

The present invention also provides use of a steroid saponin in thepreparation of a medicament for promoting the activity of an anti-cancertherapy in a subject.

The present invention also provides a method of inhibiting formationand/or growth of a tumour in a subject, the method including exposingthe subject to an anti-cancer therapy and an effective amount of asteroid saponin.

The present invention also provides use of a steroid saponin and ananti-cancer agent in the preparation of a medicament for inhibitingformation and/or growth of a tumour in a subject.

The present invention also provides a method of preventing and/ortreating a cancer in a subject, the method including exposing thesubject to an anti-cancer therapy and an effective amount of a steroidsaponin.

The present invention also provides use of a steroid saponin and ananti-cancer agent in the preparation of a medicament for preventingand/or treating cancer in a subject.

The present invention also provides a combination product including:

-   -   a steroid saponin; and    -   an anti-cancer agent;        the steroid saponin and the anti-cancer agent provided in a form        for co-administration to a subject or in a form for separate        administration to a subject.

The present invention also provides an anti-cancer composition, thecomposition including an anti-cancer agent and a steroid saponin.

The present invention also provides a method of reducing the amount ofan anti-cancer therapy provided to a subject to prevent and/or treat acancer in the subject, the method including exposing the subject to aneffective amount of a steroid saponin.

The present invention also provides use of a steroid saponin in thepreparation of a medicament for reducing the amount of an anti-cancertherapy provided to a subject to prevent and/or treat a cancer.

The present invention also provides a method of preventing and/ortreating a cancer in a subject having an increased resistance to ananti-cancer therapy, the method including exposing the subject to aneffective amount of a steroid saponin.

The present invention also provides use of a steroid saponin in thepreparation of a medicament for preventing and/or treating a cancer in asubject having an increased resistance to an anti-cancer therapy.

The present invention also provides a method of reducing resistancedeveloping in a cancerous cell to an anti-cancer therapy, the methodincluding exposing the cancerous cell to an effective amount of asteroid saponin.

The present invention also provides use of a steroid saponin in thepreparation of a medicament for reducing resistance developing in acancerous cell to an anti-cancer therapy.

The present invention also provides a method of promoting apoptosis of acancerous cell due to exposure of the cancerous cell to anti-cancertherapy, the method including exposing the cancerous cell to aneffective amount of a steroid saponin.

The present invention also provides use of a steroid saponin in thepreparation of a medicament for promoting apoptosis of a cancerous celldue to exposure of the cancerous cell to an anti-cancer therapy.

The present invention also provides a pharmaceutical compositionincluding deltonin.

The present invention also provides use of deltonin in the preparationof a medicament.

The present invention also provides a pharmaceutical compositionincluding prosapogenin A.

The present invention also provides use of prosapogenin A in thepreparation of a medicament.

The present invention also provides a pharmaceutical compositionincluding asperin.

The present invention also provides use of asperin in the preparation ofa medicament.

Various terms that will be used throughout the specification havemeanings that will be well understood by a skilled addressee. However,for ease of reference, some of these terms will now be defined.

The term “glycoside” as used throughout the specification is to beunderstood to mean a compound that contains a saccharide (sugar) moiety(monosaccharide, disaccharide or polysaccharide), linked to a triterpeneor steroid or steroid alkaloid aglycone (non-saccharide) component. Inmost circumstances, the saccharide (sugar) moiety is linked to the C-3position of the aglycone, although other linkages are contemplatedwithin the scope of the present invention. For example the furostanolglycosides, which contain a saccharide attached to the C-26 position,and spirostanol glycosides are both sub-classes of the steroid saponins.

The term “saponin” as used throughout the specification is to beunderstood to mean a glycoside including a saccharide (sugar) attachedto the aglycone, generally through the C-3 position of the aglycone.

The term “steroid saponin” as used throughout the specification is to beunderstood to mean a glycoside including one or more saccharide units(including one or more monosaccharide, disaccharide or polysaccharideunits) attached to an aglycone which does not contain a nitrogen atom.

In this regard, it will be understood that the term “steroid saponin”includes within its scope any salts or any other derivatives of thecompounds that are functionally equivalent in terms of their ability toenhance the activity of an anti-cancer therapy.

A steroid “aglycone” is also called a “genin” or “sapogenin” and theterms may be used interchangeably throughout the specification and allare to be understood to mean the non-saccharide portion of a saponinmolecule.

The term “saccharideA-(1→n)-saccharideB” as used throughout thespecification is to be understood to mean that saccharideA is linked byits C-1 to the C-n of saccharideB, n being an integer.

For example the polysaccharide with the common name “chacotriose” isα-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside.An abbreviated form of nomencalture in accordance with IUPACrecommendations used herein is Rha 2, [Rha 4], Glc.

The term “anti-cancer therapy” as used throughout the specification isto be understood to mean an anti-cancer agent, such as achemotherapeutic agent (eg cisplatin) or a biological agent (eg anantibody), or an anti-cancer treatment, such as radiotherapy.

The term “subject” as used throughout the specification is to beunderstood to mean any human or animal subject. In this regard, it willbe understood that the present invention includes within its scopeveterinary applications. For example, the animal subject may be amammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, apig, or a goat), a companion animal (eg. a dog, a cat), a laboratorytest animal (eg. a mouse, a rat, a guinea pig, a bird), an animal ofveterinary significance, or an animal of economic significance.

The term “treat”, and variants thereof as used throughout thespecification, is to be understood to mean therapeutic intervention withan effective amount of a steroid saponin. For example, the term includeswithin its scope therapeutic intervention to have one or more of thefollowing outcomes: (i) inhibit or prevent the growth of a primarytumour in a subject, including reducing the growth of the primary tumourafter resection; (ii) inhibit or prevent the growth and formation of oneor more secondary tumours in a subject; (iii) improve the lifeexpectancy of the subject as compared to the untreated state; and (iv)improve the quality of life of the subject as compared to the untreatedstate.

The term “inhibit” as used throughout the specification is to beunderstood to mean a reduction in the progress of a process, includingany one or more of the start, rate, probability, continuation ortermination of a process.

The term “cancerous cell” as used throughout the specification inrelation to cells is to be understood to mean a cell that isimmortalized and whose growth is not contact inhibited by other cells. Acancerous cell may also no longer show a dependence on exogenous growthfactors and/or anchorage dependent growth.

The term “biological system” as used throughout the specification is tobe understood to mean any multi-cellular system and includes isolatedgroups of cells to whole organisms. For example, the biological systemmay be cells in tissue culture, a tissue or organ, or an entire humansubject suffering the effects of undesired or uncontrolled growth ofcancerous cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of deltonin on tumour volume in combination with5-Fluorouracil in HT29 human prostate carcinoma cell subcutaneouslyintroduced into mice.

GENERAL DESCRIPTION OF THE INVENTION

As mentioned above, in one embodiment the present invention provides amethod of inhibiting growth of a cancerous cell, the method includingexposing the cancerous cell to an anti-cancer therapy and an effectiveamount of a steroid saponin.

The present invention is based on the finding that steroid saponins havethe ability to promote the activity of anti-cancer therapies. Thus asteroid saponin may be used in combination with an anti-cancer therapyto inhibit growth of a cancerous cell.

The cancerous cell in the various embodiments of the present inventionmay be a human or animal cell.

The cancerous cell may be a cancerous cell present in vivo or in vitro.For example, the cancerous cell may be a cancerous cell present in invitro cell culture.

In the case of a cell in vitro, the cancerous cell may be a primarycell, such as a cancerous cell isolated or derived from a tumour in asubject. Alternatively, the cancerous cell may be a cell derived from acancerous cell line. Examples of cancerous cell lines include humanmelanoma, colon adenocarcinoma (WiDr), mammary carcinoma (MCF7), mouseT-cell lymphoma (WEHI-7), mouse fibrosarcoma (WEHI-164/IC), SKMe128(melanoma), HT29 (colon), CI80-13S (ovarian), A549 (lung), DU145(prostate—hormone independent), PC3 (prostate—hormone independent),LNCap (prostate—hormone dependent), K562 (human erythroleukaemia) andMM96L (melanoma).

The cancerous cell in the various forms of the present invention mayalso be a cell present in a biological system, such as a cancerous cellpresent in vivo, including a cancerous cell that is associated with aprimary tumour and/or one or more secondary tumours in a subject.

In this regard, the term “biological system” is to be understood to meanany multi-cellular system and includes isolated groups of cells to wholeorganisms. For example, the biological system may be a tissue or organ,or an entire subject, including a subject with cancer.

Accordingly, in another embodiment the present invention provides amethod of inhibiting growth of a cancerous cell in a biological system,the method including exposing the cancerous cell to an anti-cancertherapy and an effective amount of a steroid saponin.

In the case of a cancerous cell present in a subject, the cancerous cellmay be associated for example with one or more of the following cancers:carcinoma, bladder cancer, bone cancer, brain tumours, breast cancer,cervical cancer, colorectal cancer including cancer of the colon,rectum, anus, and appendix, cancer of the oesophagus, Hodgkin's disease,kidney cancer, cancer of the larynx, leukaemia, liver cancer, lungcancer, lymphoma, melanoma, moles and dysplastic nevi, multiple myeloma,muscular cancer, non-Hodgkin's lymphoma, oral cancer, ovarian cancer,cancer of the pancreas, prostate cancer, sarcoma, skin cancer, stomachcancer, testicular cancer, teratoma, thyroid cancer, and cancer of theuterus.

In one embodiment, the anti-cancer therapy is the exposure of thecancerous cell to an anti-cancer agent, such as a chemotherapeutic agentor a biological agent.

In another embodiment, the anti-cancer therapy is exposure of thecancerous cell to an anti-cancer treatment, such as radiotherapy.

The present invention also provides use of a steroid saponin andanti-cancer agent in the preparation of a medicament for inhibitinggrowth of a cancerous cell in a subject.

As discussed above, the present invention may used to promote theactivity of an anti-cancer therapy in a subject, by exposing the subjectto a steroid saponin.

Accordingly, in another embodiment the present invention provides amethod of promoting the activity of an anti-cancer therapy in a subject,the method including exposing the subject to an effective amount of asteroid saponin.

A steroid saponin may also be used in the preparation of a medicamentfor promoting the activity of an anti-cancer agent.

Accordingly, in another embodiment the present invention provides use ofa steroid saponin in the preparation of a medicament for promoting theactivity of an anti-cancer agent in a subject.

The present invention may also be used to inhibit the formation and/orgrowth of a tumour in a subject.

Accordingly, in another embodiment the present invention provides amethod of inhibiting the formation and/or growth of a tumour in asubject, the method including exposing the subject to an anti-cancertherapy and an effective amount of a steroid saponin.

A steroid saponin and an anti-cancer agent may also be used in thepreparation of a medicament for inhibiting the formation and/or growthof a tumour in a subject.

Accordingly, in another embodiment the present invention provides use ofa steroid saponin and an anti-cancer agent in the preparation of amedicament for inhibiting growth and/or formation of tumour in asubject.

The tumour in the various embodiment of the present invention may be aprimary tumour or a secondary tumour. Thus, the present invention mayalso be used to inhibit the formation and growth of a primary tumour,and/or be used to inhibit the formation and/or growth of metastases inthe subject.

Methods for assessing the formation and/or growth of tumours are knownin the art.

The present invention may also be used to prevent and/or treat a cancerin subject.

Accordingly, in another embodiment the present invention provides amethod of preventing and/or treating a cancer in a subject, the methodincluding exposing the subject to an anti-cancer therapy and aneffective amount of a steroid saponin.

A steroid saponin and an anti-cancer agent may also be used in thepreparation of a medicament for preventing and/or treating a cancer in asubject.

Accordingly, in another embodiment the present invention provides use ofa steroid saponin and an anti-cancer agent in the preparation of amedicament for preventing and/or treating cancer in a subject.

The subject in the various embodiments of the present invention may be ahuman or animal subject.

For example, the animal subject may be a mammal, a primate, a livestockanimal (eg. a horse, a cow, a sheep, a pig, or a goat), a companionanimal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat,a guinea pig, a bird), an animal of veterinary significance, or ananimal of economic significance.

In one embodiment, the subject is a human subject.

The inhibition of growth of the cancerous cell in the variousembodiments of the present invention is any form of inhibition ofproliferation of the cell. For example, the inhibition of proliferationmay involve inhibiting the ability of a cell to begin proliferating,continue proliferating; or reducing the probability that a particularcell will begin or continue proliferating.

Inhibition of the growth of a cancerous cell in the various embodimentsof the present invention may be assessed by a method known in the art.

For example, for a cancerous cell in vitro, the growth of the cancerouscell may be determined by a suitable proliferation assay, or by a methodfor assessing the extent of incorporation of tritiated thymidine intocellular DNA over a given period of time.

For a cancerous cell present in vivo, the growth of the cancerous cellmay be determined for example by a suitable imaging method known in theart.

As discussed previously herein, the anti-cancer therapy may be exposureto an anti-cancer agent and/or exposure to an anti-cancer treatment.

In one embodiment, the anti-cancer agent is an agent that promotesapoptosis in a cell upon exposure of the agent to the cell. Methods fordetermining the ability of an agent to promote apoptosis are known inthe art.

In one specific embodiment, the anti-cancer agent inhibits the activityof an inhibitor of apoptosis in the cancerous cell, such as one or moreof sunrivin, XIAP, Bcl-2 or Bcl-XL.

In another embodiment, the anti-cancer agent is a chemotherapeuticagent, such as an alkylating agent, including BCNU (carmustine),bisulfan, CCNU (lomustine), chlorambucil, cisplatin, oxiplatin, melphan,mitomycin C, and thio-tepa; antimitotic agents including taxol(paclitaxel), docetaxel, vinblastine sulphate, and vincristine sulphate;topoisomerase inhibitors including doxorubicin, daunorubicin, m-AMSA(amsacrine), mitoxantrone, and VP-16 (etoposide); RNA/DNAantimetabolites including 5-fluorouracil and methotrexate; DNAantimetabolites including Ara-C (cytarabine), hydroxyurea(hydroxycarbamide), and thioguanine (tioguanine).

In another embodiment the anti-cancer agent is a cellular processtargeting-agent such as imatinib mesylate, trastuzumab, and gefitinib.

Details of administration routes, doses, and treatment regimes ofanti-cancer agents are known in the art, for example as described in“Cancer Clinical Pharmacology” (2005) ed. By Jan H. M. Schellens, HowardL. McLeod and David R. Newell, Oxford University Press.

Saponins are conventionally divided into three major classes: (i)triterpene glycosides; (ii) steroidal glycosides; and (iii) steroidalalkaloid glycosides. They all have in common the attachment of one ormore sugar units to the aglycone, generally at the C-3 position. Steroidsaponins are generally as described in Hostettmann K and Marston A(2005). Chemistry & pharmacology of natural products: Saponins,Cambridge University Press.

As discussed previously herein, steroid saponins do not contain anitrogen atom in the aglycone moiety.

It will be appreciated that the steroid saponin in the variousembodiments of the present invention include naturally occurring steroidsaponins and non-naturally occurring steroid saponins (ie chemicallysynthesized steroid saponins). In addition, it will also be appreciatedthat the steroid saponin in the various embodiments of the presentinvention also includes pro-drugs of the steroid saponin, derivatives ofsteroid saponins, including for example, any esters, ketones, carboxylicacids, salts, substituted forms, halogenated forms or other heteroatomcontaining forms, unsaturated forms, or any other functional derivative.

The saccharide portion of the steroid saponins in the variousembodiments of the present invention may include one or more saccharideunits, such as a monosaccharide, a disaccharide unit or a polysaccharideunit.

It will also be appreciated that the steroid saponin of the variousembodiments of the present invention may also include an aglycone with asaccharide attached at one or more positions of the aglycone moiety.

In one embodiment, the steroid saponin includes a saccharide attached toa single position of the sapogenin component of the steroid saponin.

As discussed above, the saccharide unit may be a monosaccharide, adisaccharide or a polysaccharide. The saccharide may be composed of asuitable monosaccharide, such as D-glucose (Glc), L-rhamnose (Rha),D-galactose (Gal), D-glucuronic acid (GlCA), D-xylose (Xyl), L-arabinose(Ara), D-fucose (Fuc), D-galacturonic acid (GalA). The saccharide unitmay also be a substituted sugar, such as an amino sugar, a sulphatedsugar, an acylated sugar, a N-acylated sugar, and functional derivativesof any of the aforementioned monosaccharides.

Similarly, a disaccharide may be any combination of two monosaccharides,as described above.

The polysaccharides in the various embodiments of the present inventionmay be linear or branched, and include any combination of two or moremonosaccharide, including the monosaccharide described previouslyherein.

In one embodiment, the polysaccharide is composed of 1 to 6monosaccharide units.

In this regard, and as described previously herein, polysaccharides aregenerally described in the context of the arrangement of the componentmonosaccharides.

In one embodiment, the saccharide of the steroid saponin is composed of1 monosaccharide unit. An example of a monosaccharide is glucose withthe chemical name β-D-glucopyranoside, which when attached to theaglycone diosgenin via the C-3 position, has the common name of“trillin.”

In another embodiment, the saccharide of the steroid saponin is composedof 2 monosaccharide units (ie a disaccharide). An example of adisaccharide is Rha 2, Glc with the chemical nameα-L-rhamnopyranosyl(1→2)-β-D-glucopyranoside, which when attached to theaglycone diosgenin via the C-3 position, has the common name of“prosapogenin A.”

In another embodiment, the saccharide of the steroid saponin is composedof 3 saccharide units (ie a trisaccharide). Chacotrioside is a commonexample of a trisaccharide unit, where the glycosyl group of threesaccharides comprises two rhamnose units linked to a glucose unit whichin turn is linked via a glycosidal linkage to the C-3 position of asapogenin. Chacotriose isα-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1-4)]-β-D-glucopyranosidewhereas an abbreviated form of nomencalture in accordance with IUPACrecommendations used herein is Rha 2, [Rha 4], Glc.

Similarly solatrioside is a glycosyl group of three saccharidescomprising one rhamnose unit and a non-rhamnose saccharide unit, eachlinked to a third saccharide unit, which is in turn linked via aglycosidal linkage to the C-3 position of a sapogenin.

An example of a tetrasaccharide is [Rha 4, Rha 4], Rha 2, Glc with thechemical name [α-L-rhamnopyranosyl(1→4)-α-L-rhamnopyranosyl(l4)]-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranoside, which when attached tothe aglycone diosgenin via the C-3 position has the common name of“asperin.”

Another example of a tetrasaccharide is Glc 4, [Xyl 3], Rha 2, Ara, withthe chemical nameβ-D-glucopyranosyl(1→4)-[β-D-xylopyranosyl-(1→3)]-α-L-rhamnopyranosyl(1→2)-α-L-arabinoside.

As discussed previously herein, steroid saponins do not contain anitrogen atom in the aglycone moiety.

Accordingly, it will be appreciated that the steroid saponin in thevarious embodiments of the present invention will not contain a nitrogengroup in the sapogenin moiety, such as not containing a nitrogen in theE and/or F rings of the sapogenin.

In one embodiment, the steroid saponin in the various embodiments of thepresent is based on a sapogenin with the chemical formula I or II asfollows:

wherein

R₁, R₂, R₄, R₆, R₇, R₁₁, R₁₂, R₁₄, R₁₅ and R₁₇ are independently H, OH,═O, pharmacologically acceptable ester groups or pharmacologicallyacceptable ether groups;R₅ is H when C-5,C-6 is a single bond, and nothing when C-5,C-6 is adouble bond;A is either 0 concurrently with B being CH₂, or B is O concurrently withA being CH₂;R_(27A) is H concurrently with R_(27B) being CH₃, or R_(27A) is CH₃concurrently with R_(27B) being H;R₃ comprises a glycosyl group linked through the oxygen atom to thesteroidal sapogenin at C-3; or a pharmaceutically acceptable salt, orderivative thereof.

whereinR₁, R₂, R₄, R₆, R₇, R₁₁, R₁₂, R₁₄, R₁₅ and R₁₇ are independently H, OH,═O, pharmacologically acceptable ester groups or pharmacologicallyacceptable ether groups;R₅ is H when C-5,C-6 is a single bond, and nothing when C-5,C-6 is adouble bond;R₂₂ is either a hydroxyl or an alkoxyl group when C-20, C-22 is a singlebond, or nothing when C-20, C-22 is a double bond;R_(27A) is H concurrently with R_(27B) being CH₃, or R_(27A) is CH₃concurrently with R_(27B) being H;R₂₈ is H or a saccharide; or a pharmaceutically acceptable salt, orderivative thereof;R₃ comprises a glycosyl group linked through the oxygen atom to thesteroidal sapogenin at C-3; or a pharmaceutically acceptable salt, orderivative thereof.

Examples of steroid sapogenins include spirostanol aglycones such asdiosgenin, yamogenin (neodiosgenin), yuccagenin, sarsasapogenin,tigogenin, smilagenin, hecogenin, gitogenin, convallamarogenin,neoruscogenin, and solagenin; and furostanol aglycones such asprotodiosgenin, pseudoprotodiosgenin, methyl protodiosgenin,protoyamogenin, and methyl protoyamogenin.

In one embodiment, the steroid saponin is a chacotrioside-steroidsaponin or a solatrioside-steroid saponin.

Examples of chacotrioside-steroid saponins include “dioscin” whichconsists of the sapogenin “diosgenin” linked through the C-3 position tochacotriose, diosgenin linked through the C-3 position to anotherchacotrioside, tigogenin linked through the C-3 position to achacotrioside, sarsasapogenin linked through the C-3 position to achacotrioside, smilagenin linked through the C-3 position to achacotrioside, yuccagenin linked through the C-3 position to achacotrioside, and yamogenin linked through the C-3 position to achacotrioside.

Examples of solatrioside steroid saponins include “gracillin”, which isdiosgenin linked through the C-3 position to the solatrioside (Rha 2,[Glc 3], Glc); “deltonin” (diosgenin linked through the C-3 position tothe solatrioside Rha 2, [Glc 4], Glc); diosgenin linked through the C-3position to solatriose (Rha 2, [Glc 3], Gal) [in this context, diosgeninlinked to (Rha 2, [Glc 3], Gal) is termed ‘diosgenin solatriose’ ];diosgenin linked through the C-3 position to another solatrioside;tigogenin linked through the C-3 position to a solatrioside;sarsasapogenin linked through the C-3 position to a solatrioside;smilagenin linked through the C-3 position to a solatrioside; yuccageninlinked through the C-3 position to a solatrioside, and yamogenin linkedthrough the C-3 position to a solatrioside.

Simple monosaccharide steroid saponins are widespread in the plantkingdom. The monosaccharide is generally linked to the aglycone throughthe C-3 position and examples include “trillin,” which is diosgeninlinked through the C-3 position to glucose. Other sapogenins linked toglucose through the C-3 position include sarsasapogenin, rhodeasapogeninand yamogenin. Some sapogenins are linked through the C-3 position toanother monosaccharide such as arabinose eg, yonogenin and convallageninor linked through the C-3 position to galactose and so forth.

Examples of disaccharide steroid saponins include sarsasapogenin linkedthrough the C-3 position to for example (Xyl 2, Gal); (Glc 2, Glc); (Glc3, Glc); smilagenin linked through the C-3 position to (Glc 2, Glc);(Glc 2, Gal); samogenin, tigogenin, gitogenin, alliogenin, ruscogenin,pennogenin, cepagenin and diosgenin linked through the C-3 position to(Rha 2, Glc).

The diosgenin glycosides from Dioscorea species are of great commercialinterest as starting materials for steroid hormones. Glycosides ofdiosgenin and its C-25 isomer yamogenin are among the most frequentlydocumented spirostanol saponins. Examples of naturally occurring steroidspirostanol sapogenins with a C-5,C-6 double bond in the B-ring arelisted in Table 1:

TABLE 1

R₁ R₂ R₁₂ R_(27A) R_(27B) Diosgenin H H H H CH₃ Yamogenin H H H CH₃ HYuccagenin H OH H H CH₃ Gentrogenin H H ═O H CH₃ Ruscogenin OH H H H CH₃

Examples of naturally occurring steroid spirostanol sapogenins with aC-5, C-6 single bond in the B-ring are listed in Table 2:

TABLE 2

R₂ H₅ R₆ R₁₂ R₁₅ R₂₈ R₂₉ Smilagenin H β H H H H CH₃ Tigogenin H α H H HH CH₃ Sarsasapogenin H β H H H CH₃ H Gitogenin OH β H H H H CH₃Hecogenin H α H ═O H H CH₃ Chlorogenin H α OH(α) H H H CH₃ DigitogeninOH(α) α H H OH(β) H CH₃ Digalogenin H α H H OH(β) H CH₃

Examples of naturally occurring steroid furostanol sapogenins of theprotospirostane-type with a C-5,C-6 double bond in the B-ring and aC-20,C-22 single bond in the E-ring, are listed in Table 3:

TABLE 3

R₂₂ Protodiosgenin H Methyl protodiosgenin CH₃

An example of a naturally occurring steroid furostanol sapogenin of theprotospirostane-type with a C-5, C-6 single bond in the B-ring and aC-20, C-22 single bond in the E-ring, is prototigogenin.

An example of a naturally occurring steroid furostanol sapogenin of thepseudospirostane-type with a C-5, C-6 double bond in the B-ring and aC-20,C-22 double bond in the E-ring is pseudodiosgenin.

An example of a naturally occurring steroid furostanol sapogenin of thepseudoprotospirostane-type with a C-5,C-6 double bond in the B-ring anda C-20,C-22 double bond in the E-ring is pseudoprotodiosgenin.

In one embodiment, the steroid saponin is the sapogenin diosgenin linkedthrough the C-3 position to one or more monosaccharide units.

In another embodiment, the steroid saponin is dioscin or gracillin,where dioscin is the sapogenin diosgenin linked through the C-3 positionto chacotriose (Rha 2, [Rha 4], Glc) and gracillin is diosgenin linkedthrough the C-3 position to the solatrioside (Rha 2, [Glc 3], Glc).

In another embodiment, the steroid saponin is diosgenin linked throughthe C-3 position to solatriose (Rha 2, [Glc 3], Gal). In this context,diosgenin linked to (Rha 2, [Glc 3], Gal) is termed ‘diosgeninsolatriose’.

In another embodiment, the steroid saponin is the sapogenin diosgeninlinked through the C-3 position to a saccharide.

In another embodiment, the steroid saponin is the sapogenin tigogenin,linked through the C-3 position to a saccharide.

In another embodiment, the steroid saponin is the sapogeninsarsasapogenin, linked through the C-3 position to a saccharide.

In another embodiment, the steroid saponin is the sapogenin smilagenin,linked through the C-3 position to a saccharide.

In another embodiment, the steroid saponin is the sapogenin yuccagenin,linked through the C-3 position to a saccharide.

In another embodiment, the steroid saponin is the sapogenin yamogenin,linked through the C-3 position to a saccharide.

In one specific embodiment, the steroid saponin is selected from thegroup consisting of deltonin (diosgenin Rha2, [Glc4], Glc), dioscin(diosgenin Rha2, [Rha4], Glc), prosapogenin A (diosgenin Rha2, Glc) andasperin (diosgenin [Rha 4, Rha 4], Rha 2, Glc).

In the case of deltonin, prosapogenin A and asperin, any one of thesesteroid saponins may be prepared in a pharmaceutical composition.

Accordingly, such steroid saponins may be used in the preparation of amedicament.

Such a medicament may be used for one or more of inhibiting growth of acancerous cell; inhibiting formation and/or growth of a tumour;preventing and/or treating a cancer, including a cancer having increasedresistance to an anti-cancer therapy; promoting the activity of ananti-cancer therapy; reducing the amount of an anti-cancer therapyprovided to a subject; promoting apoptosis of a cancerous cell due toexposure of the cell to an anti-cancer therapy; reducing resistancedeveloping in a cancerous cell to an anti-cancer therapy.

As discussed previously herein, the steroid saponin in the variousembodiments of the present invention may be obtained from naturalsources, manufactured from synthesis processes, or as partial synthesisor modification applied to naturally occurring compounds orintermediates.

The extraction, isolation and identification of steroid saponins in thevarious embodiments of the present invention may be achieved by methodsknown in the art.

For example, some steroid saponins may be produced from plant sources.Other sources of steroid saponins may be readily obtained from theliterature, for example as described in Hostettmann K and Marston A(2005). Chemistry & pharmacology of natural products: Saponins.Cambridge University Press, chapters 1-3 and 6. Common names of steroidsaponins have been used in accordance with the above text and theDictionary of Natural Products, Chapman and Hall, CRC, (2004).

Methods are known in the art for exposing cancerous cells in vitro andin vivo to anti-cancer agents and anti-cancer treatments.

Methods are also known in the art for exposing a steroid saponin to acancerous cell in vitro and in vivo.

A suitable method for exposing a steroid saponin to the cancerous cellin vitro is by direct exposure of the steroid saponin to the cancerouscell.

In the case of a cancerous cell in a subject, a suitable method ofexposing the cancerous cell to the steroid saponin is by administrationof the saponin to the subject.

Effective amounts of anti-cancer agents, and effective levels ofanti-cancer treatments, are known in the art. Methods for exposingcancerous cells in vitro and in vivo to anti-cancer agents andtreatments are known in the art.

The effective amount of the steroid saponin to be exposed to thecancerous cell in the various embodiments of the present invention isnot particularly limited. Generally an effective concentration of thesteroid saponin will be in the range from 0.1 μM to 20 μM.

In the case of the use of a steroid saponin to enhance the activity ofan anti-cancer agent in a subject, the steroid saponin and theanti-cancer agent may be separately administered to the subject in asuitable form, or alternatively, be co-administered to the subject in asuitable form.

For example, the steroid saponin and the anti-cancer agent may beincluded in a combination product for separate or co-administration to asubject.

Accordingly, in another embodiment the present invention provides acombination product including a steroid saponin and an anti-canceragent, the steroid saponin and the anti-cancer agent provided in a formfor co-administration to a subject or in a form for separateadministration to a subject.

The combination product is suitable for, for example, inhibiting thegrowth of cancerous cells, for inhibiting tumour formation and growth(primary and/or secondary tumours), and for preventing and/or treating acancer.

The components of the combination product may be packaged separately ortogether in suitably sterilized containers such as ampoules, bottles, orvials, either in multi-dose or in unit dosage forms. The containers aretypically hermetically sealed. Methods are known in the art for thepackaging of the components.

As discussed previously herein, co-administration of the steroid saponinand an anti-cancer agent can be sequential or simultaneous and generallymeans that the agents are present in the subject during a specified timeinterval. Typically, if a second agent is administered within thehalf-life of the first agent, the two agents are consideredco-administered.

An appropriate dosage regime for the administration of the steroidsaponin may be chosen by a person skilled in the art. For example, theadministration of the steroid saponin to the subject may be prior to,concurrently with, or after exposure of the subject to the anti-cancertherapy.

In one embodiment, the steroid saponin is administered to a subjectconcurrently with administration of an anti-cancer agent to a subject,or concurrently with exposure of the subject to an anti-cancertreatment.

In one specific embodiment, the steroid saponin and the anti-canceragent may be included in a single composition for administration to asubject.

Accordingly, in another embodiment the present invention provides apharmaceutical composition including a steroid saponin and ananti-cancer agent. In one embodiment, the composition is an anti-cancercomposition.

Accordingly, in another embodiment the present invention provides ananti-cancer composition including an anti-cancer agent and a steroidsaponin.

The composition may be used, for example, for inhibiting growth of acancerous cell in vitro or in vivo.

The composition may also be used for inhibiting tumour formation andgrowth (primary and/or secondary tumours), and for preventing and/ortreating a cancer in a subject.

The effective amount of the steroid saponin, and the effective amount ofan anti-cancer agent or an anti-cancer treatment, to be administered tothe subject is not particularly limited, so long as it is within such anamount and in such a form that generally exhibits a useful ortherapeutic effect. The term “therapeutically effective amount” is thequantity which, when administered to a subject in need of treatment,improves the prognosis and/or health state of the subject. The amount tobe administered to a subject will depend on the particularcharacteristics of one or more of the cancerous cell for which growth isto be inhibited, the cancer being treated, the mode of administration,and the characteristics of the subject, such as general health, otherdiseases, age, sex, genotype, and body weight. A person skilled in theart will be able to determine appropriate dosages depending on these andother factors.

In this regard, details of administration routes, doses, and treatmentregimes of anti-cancer agents and radiotherapy treatment are known inthe art, for example as described in “Cancer Clinical Pharmacology”(2005) ed. By J. H. M. Schellens, H. L. McLeod and D. R. Newell, OxfordUniversity Press; and “Cancer and its management” (2005). Fifth Editionby R. Souhami and J. Tobias, Blackwell Publishing.

As discussed previously herein, administration and delivery of thecompositions according to the present invention may be by, for example,the intravenous, intraperitoneal, subcutaneous, intramuscular, oral, ortopical route, or by direct injection into the site of a primary tumourprior to, during or following additional forms of treatment includingsurgery. The mode and route of administration in most cases will dependon the type of tumour being treated.

The dosage form, frequency and amount of dose will depend on the modeand route of administration. Typically an injectable composition will beadministered in an amount of between 5 mg/m² and 500 mg/m², generallybetween 10 mg/m² and 200 mg/m².

Typically an orally administered composition will be administered in anamount of between 5 mg and 5 g, preferably between 50 mg and 1 g.

For example, effective amounts of the steroid saponin typically rangebetween about 0.1 mg/kg body weight per day and about 1000 mg/kg bodyweight per day, and in one form between 1 mg/kg body weight per day and100 mg/kg body weight per day.

As described above, the administration of a composition including asteroid saponin may also include the use of one or more pharmaceuticallyacceptable additives, including pharmaceutically acceptable salts, aminoacids, polypeptides, polymers, solvents, buffers, excipients,preservatives and bulking agents, taking into consideration theparticular physical, microbiological and chemical characteristics of thesteroid saponins to be administered.

For example, the steroid saponin can be prepared into a variety ofpharmaceutical acceptable compositions in the form of, e.g., an aqueoussolution, an oily preparation, a fatty emulsion, an emulsion, alyophilised powder for reconstitution, etc., and can be administered asa sterile and pyrogen free intramuscular or subcutaneous injection or asinjection to an organ, or as an embedded preparation or as atransmucosal preparation through nasal cavity, rectum, uterus, vagina,lung, etc. The composition may be administered in the form of oralpreparations (for example solid preparations such as tablets, caplets,capsules, granules or powders; liquid preparations such as syrup,emulsions, dispersions or suspensions).

Compositions containing the steroid saponin may also contain one or morepharmaceutically acceptable preservative, buffering agent, diluent,stabiliser, chelating agent, viscosity-enhancing agent, dispersingagent, pH controller, or isotonic agent. These excipients are well knownto those skilled in the art.

Examples of suitable preservatives are benzoic acid esters ofpara-hydroxybenzoic acid, phenols, phenylethyl alcohol or benzylalcohol. Examples of suitable buffers are sodium phosphate salts, citricacid, tartaric acid and the like. Examples of suitable stabilisers areantioxidants such as alpha-tocopherol acetate, alpha-thioglycerin,sodium metabisulphite, ascorbic acid, acetylcysteine,8-hydroxyquinoline, and chelating agents such as disodium edetate.Examples of suitable viscosity enhancing agents, suspending,solubilizing or dispersing agents are substituted cellulose ethers,substituted cellulose esters, polyvinyl alcohol, polyvinylpyrrolidone,polyethylene glycols, carbomer, polyoxypropylene glycols, sorbitanmonooleate, sorbitan sesquioleate, polyoxyethylene hydrogenated castoroil 60.

Examples of suitable pH controllers include hydrochloric acid, sodiumhydroxide, buffers and the like. Examples of suitable isotonic agentsare glucose, D-sorbitol or D-mannitol, sodium chloride.

The administration of the steroid saponin in the various embodiments ofthe present invention may also be in the form of a compositioncontaining a pharmaceutically acceptable carrier, diluent, excipient,suspending agent, lubricating agent, adjuvant, vehicle, delivery system,emulsifier, disintegrant, absorbent, preservative, surfactant, colorant,glidant, anti-adherent, binder, flavorant or sweetener, taking intoaccount the physical, chemical and microbiological properties of thesteroid saponin being administered.

For these purposes, the composition may be administered for exampleorally, parenterally, by inhalation spray, adsorption, absorption,topically, rectally, nasally, bucally, vaginally, intraventricularly,via an implanted reservoir in dosage formulations containingconventional non-toxic pharmaceutically-acceptable carriers, or by anyother convenient dosage form. The term parenteral as used hereinincludes subcutaneous, intravenous, intramuscular, intraperitoneal,intrathecal, intraventricular, intrasternal, and intracranial injectionor infusion techniques.

When administered parenterally, the composition will normally be in aunit dosage, sterile, pyrogen free injectable form (solution, suspensionor emulsion, which may have been reconstituted prior to use) which ispreferably isotonic with the blood of the recipient with apharmaceutically acceptable carrier. Examples of such sterile injectableforms are sterile injectable aqueous or oleaginous suspensions. Thesesuspensions may be formulated according to techniques known in the artusing suitable vehicles, dispersing or wetting agents, complexingagents, polymers, solubility aids and suspending agents. The sterileinjectable forms may also be sterile injectable solutions or suspensionsin non-toxic parenterally acceptable diluents or solvents, for example,as solutions in 1,3-butanediol. Among the pharmaceutically acceptablevehicles and solvents that may be employed are water, ethanol, glycerol,saline, dimethylsulphoxide, N-methylpyrrolidone, dimethylacetamide,Ringer's solution, dextrose solution, isotonic sodium chloride solution,and Hanks' solution. In addition, sterile, fixed oils are conventionallyemployed as solvents or suspending mediums. For this purpose, any blandfixed oil may be employed including synthetic mono- or di-glycerides,corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyloleate, isopropyl myristate, and oleic acid and its glyceridederivatives, including olive oil and castor oil, especially in theirpolyoxyethylated versions, are useful in the preparation of injectables.These oil solutions or suspensions may also contain long-chain alcoholdiluents or dispersants.

The carrier may also contain additives, such as substances that enhancesolubility, isotonicity, and chemical stability, for exampleanti-oxidants, buffers and preservatives.

In addition, the composition containing the steroid saponin may be in aform to be reconstituted prior to administration. Examples includelyophilisation, spray drying and the like to produce a suitable solidform for reconstitution with a pharmaceutically acceptable solvent priorto administration.

Compositions may include one or more buffer, bulking agent, isotonicagent and cryoprotectant and lyoprotectant. Examples of excipientsinclude, phosphate salts, citric acid, non-reducing sugars such assucrose or trehalose, polyhydroxy alcohols, amino acids, methylamines,and lyotropic salts are preferred to the reducing sugars such as maltoseor lactose.

When administered orally, the steroid saponin will usually be formulatedinto unit dosage forms such as tablets, caplets, cachets, powder,granules, beads, chewable lozenges, capsules, liquids, aqueoussuspensions or solutions, or similar dosage forms, using conventionalequipment and techniques known in the art. Such formulations typicallyinclude a solid, semisolid, or liquid carrier. Exemplary carriersinclude excipients such as lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoabutter, oil of theobroma, alginates, tragacanth, gelatin, syrup,substituted cellulose ethers, polyoxyethylene sorbitan monolaurate,methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesiumstearate, and the like.

A tablet may be made by compressing or moulding the steroid saponinoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, surfaceactive, or dispersing agent. Moulded tablets may be made by moulding ina suitable machine, a mixture of the powdered active ingredient and asuitable carrier moistened with an inert liquid diluent.

The administration of the steroid saponin agent may also utilizecontrolled release technology.

For topical administration, the composition of the present invention maybe in the form of a solution, spray, lotion, cream (for example anon-ionic cream), gel, paste or ointment. Alternatively, the compositionmay be delivered via a liposome, nanosome, ribosome, or nutri-diffuservehicle. Topical administration may be used for the treatment of cancerssuch as melanomas.

It will be appreciated that in the case of the pharmaceuticalcomposition also including an anti-cancer agent, similarlyconsiderations as described above apply to the formulation of thecomposition.

The present invention may also be used to reduce the amount of ananti-cancer agent or treatment provided to a subject to prevent and/ortreat a cancer.

In this regard, the ability of a steroid saponin to increase the levelof activity of the anti-cancer therapy can be used to reduce the dose ofthe anti-cancer therapy exposed to a subject to achieve a desired levelof treatment.

Accordingly, in another embodiment the present invention provides amethod of reducing the amount of an anti-cancer therapy provided to asubject to prevent and/or treat a cancer in the subject, the methodincluding exposing the subject to an effective amount of a steroidsaponin.

The present invention also provides the use of a steroid saponin in thepreparation of a medicament for reducing the amount of an anti-cancertherapy provided to a subject to prevent and/or treat a cancer.

Accordingly, in another embodiment the present invention provides use ofa steroid saponin in the preparation of a medicament for reducing theamount of an anti-cancer therapy provided to a subject to prevent and/ortreat a cancer.

The present invention may also be used to promote apoptosis of acancerous cell due to exposure of the cell to an anti-cancer agent. Forexample, the steroid saponin may be used to promote apoptosis of a cellexposed to a chemotherapeutic agent.

Accordingly, in another embodiment the present invention provides amethod of promoting apoptosis of a cancerous cell due to exposure of thecancerous cell to an anti-cancer therapy, the method including exposingthe cancerous cell to an effective amount of a steroid saponin.

The present invention also provides the use of a steroid saponin in thepreparation of a medicament for promoting apoptosis of a cancerous celldue to exposure of the cancerous cell to an anti-cancer therapy.

Accordingly, in another embodiment the present invention provides use ofa steroid saponin in the preparation of a medicament for promotingapoptosis of a cancerous cell due to exposure of the cancerous cell toan anti-cancer therapy.

The present invention may also be used to reduce the level of resistanceof a cancerous cell to an anti-cancer agent.

For example, exposure of cancerous cells to an anti-cancer agent, suchas a chemotherapeutic drug, leads to an increased level of resistance ofthe cell to the chemotherapeutic drug. Ultimately, this may lead to thecancer developing multi-drug resistance.

Accordingly, in another embodiment the present invention provides amethod of reducing resistance developing in a cancerous cell to ananti-cancer therapy, the method including exposing the cancerous cell toan effective amount of a steroid saponin.

The present invention also provides use of the steroid saponin in thepreparation of a medicament for reducing resistance developing in acancerous cell to an anti-cancer therapy.

Accordingly, in another embodiment the present invention also providesuse of a steroid saponin in the preparation of a medicament for reducingresistance developing in a cancerous cell to an anti-cancer therapy.

The present invention also provides use of the steroid saponin and theanti-cancer agent in the preparation of a medicament to reduce the levelof resistance of a cancerous cell to an apoptotic agent.

Accordingly, in another embodiment the present invention provides use ofa steroid saponin in the preparation of a medicament for reducing thelevel of resistance of a cancerous cell to an anti-cancer agent.

Methods for the preparation of pharmaceutical compositions are known inthe art, for example as described in Remington's PharmaceuticalSciences, 18th ed., 1990, Mack Publishing Co., Easton, Pa.; U.S.Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton,Pa.; and M. E. Aulton, Pharmaceutics, The Science of Dosage Form Design,2nd ed., Churchill Livingstone, Edinburgh, 2002.

Therapeutic delivery of biomolecules is generally as described inBladon, C. (2002) “Pharmaceutical Chemistry: Therapeutic Aspects ofBiomolecules” John Wiley & Sons Ltd.

Description of Specific Embodiments

Reference will now be made to experiments that embody the above generalprinciples of the present invention. However, it is to be understoodthat the following description is not to limit the generality of theabove description.

Example 1 General Reagents and Methods (i) Steroid Saponins andAnti-Cancer Agents

Diosgenin, dioscin: diosgenin Rha2, [Rha4], Glc and deltonin: diosgeninRha2, [Glc4], Glc were obtained commercially from Ningbo HanpharmBiotech Co Ltd, and gracillin from ChromaDex, and trillin from AktinChemicals.

Prosapogenin A: diosgenin Rha2, Glc was synthesised in accordance withthe method described by Li et al Carbohydr. Res., (2001) 331, 1-7.Dioscin and prosapogenin A were also isolated from Paris polyphylla.

Steroid saponins were dissolved in dimethylsulphoxide (DMSO) to produce10 mM or 1 mM stock solutions from which further dilutions were preparedas required for individual experiments.

Cisplatin, docetaxel, paclitaxel, doxorubicin, vincristine and imitanibwere obtained from commercial sources and stored either at 4° C. or −20°C. as required. These were: paclitaxel (anzatax injection (Faulding);vincristine sulphate (Sigma); doxorubicin HCl (Sigma); docetaxel(Sigma); cisplatin (Sigma) and imatinib mesylate (Novartis Glivec).

Chemotherapeutic agents were prepared at stock concentrations asrequired (determined for each assay) in the appropriate diluent: DMSO,sterile water or saline. DMSO solution alone was used as the negativecontrol.

(ii) Cells

Human cancer cell types were: A549 (lung); CI80-13S (Ovarian); HT29(colon); MCF7 (breast); PC3 (prostate); DU145 (prostate, hormoneindependent); LNCap (hormone dependant); K562 (leukaemia). Mouse cancercell type was: B16 (melanoma).

Cancer cells were seeded the day before application of drug intriplicate or quadruplicate in 96-well plates, allowed to grow in thepresence of drug for 6 days before cell growth relative to untreatedcontrol wells was determined with a dye assay.

(iii) Cell Culture

Cells were seeded in triplicate or quadruplicate at 3-4,000 permicrotitre well in 90 μl of RPMI culture medium/10% foetal calfserum/penicillin, streptomycin mix, treated with 10 μl of drug (preparedin a dilution plate at 10× concentration required), and allowed to growuntil the controls were nearly confluent (6 days).

SRB: Plates were washed with PBS, fixed with methylated spirits, washedwith tap water and stained with 50 μL/well of SRB solution(sulforhodamine, 0.4% in 1% acetic acid), followed by washing with tapwater and 1% acetic acid, solubilisation in Tris and absorbance read at564 nm in an ELISA reader.

MTS: 10 μl of MTS solution was added/well of cells, plates allowed toincubate for 1-4 hours at 37° C. until development of a dark browncolour. 10 μl of 10% SDS was then added/well to disperse the cells.Assay plates were then centrifuged at 2000 rpm for 15 minutes andabsorbance read at 490 nm in an Elisa reader.

Data was collected using ELISA plate reader software—SOFTmax PRO3.1.2,then imported into EXCEL. The mean and SD of replicates were calculatedas a % of control, after subtraction of the blank value (wells with nocells). A graph was plotted of % control vs dose of agent and IC₅₀ (halfmaximal inhibitory concentration) values determined.

Alternatively, cells were seeded into 2 mL wells and following 24 hourgrowth were treated with steroid saponin at 0.1, 0.5 & 1.0 μM. Cellswere harvested after 24, 48 and 72 hour incubation with the drug. Ateach time point the cells were counted, pelleted by centrifugation (5minutes, 1500 rpm, RT), resuspended in 1 mL of PBS & vortexed gently. 2mL of ice cold methanol was added and the cells vortexed. Once cellsfrom all time points were collected, each sample was centrifuged at12000 rpm for 4-5 minutes, resuspended in 400 μl of PBS and 100 μl of 5×propidium iodide (PI) stain (see below) was added. Samples werevortexed, and filtered through a nylon filter prior to flow cytometryanalysis at 488 nm. Relative DNA contents of the cell subpopulationswere represented as histograms, with 20,000 cells typically analysed foreach sample.

Example 2 Determination of IC₅₀ Values for Steroid Saponins andAnti-Cancer Agents

The following cancer cell lines were used:

A549—lungHT29—colonMCF7—breastPC3—prostate (hormone independent)DU145—prostate (hormone independent)LNCap—prostate (hormone dependent)K562—human erythroleukemia

The following steroid saponins were assayed for inhibition of cancercells lines when used as single agents:

Dioscin: diosgenin Rha2, [Rha4], GlcDeltonin: diosgenin Rha2, [Glc4], GlcProsapogenin A: diosgenin Rha2, Glc

The following chemotherapeutic drugs and molecular targeting agents wereassayed for inhibition of cancer cells when used as single agents:

Chemotherapeutic agents:

Cisplatin Docetaxel Paclitaxel Doxorubicin Vincristine

Molecular targeting agent:

Imatinib

The steroid saponins and the above-listed anti-cancer agents weredissolved in DMSO and diluted with culture medium into the requiredsolutions for each application. DMSO solution alone was used as thenegative control.

Tumour cells were seeded in duplicate in 96-well plates at 2-5,000 permicrotitre well containing RPMI culture medium/10% foetal calf serum.The cells were allowed to grow until the controls were nearly confluentafter 5-6 days. Plates were then washed with PBS, fixed in ethanol andstained with 50 μL/well of SRB solution (sulforhodamine, 0.4% in 1%acetic acid), followed by washing with 1% acetic acid and solubilisationin Tris. The absorbance was read at 564 nm using an ELISA reader.

The IC₅₀, or concentration required to inhibit cell growth by 50%, wasdetermined from the percentage inhibition versus concentration datausing the probit calculation methods of Finney (Finney D J (1971).Probit Analysis. 3^(rd) Edition. Cambridge University Press).

The following concentrations described in Table 4 were used for the 8cells of the 12×8 ELISA plates:

TABLE 4 Cell concentration (μM) ELISA Cell 1 2 3 4 5 6 7 8 Cisplatin 4 21 0.5 0.25 0.125 0.0625 0 Docetaxel 0.006 0.003 0.0015 0.00075 0.0003750.000188 0.000094 0 Paclitaxel 0.0033 0.00167 0.00083 0.00042 0.000210.000104 0.000052 0 Doxorubicin 0.2 0.1 0.05 0.025 0.0125 0.00625 0.00310 Vincristine 0.02 0.01 0.005 0.0025 0.00125 0.000625 0.00031 0 Imatinib0.72 0.36 0.18 0.09 0.045 0.0225 0.0112 0

The following IC₅₀ values in Table 5 were estimated from the inhibitiondata:

TABLE 5 IC₅₀ (μM) DU145 LNCap MCF7 HT29 PC3 A549 K562 Cisplatin 0.540.70 0.65 0.93 1.4 1.2 0.82 Docetaxel 0.00015 0.000026 0.000039 0.000510.00009 0.00027 0.00042 Paclitaxel 0.0027 0.0012 0.0011 >0.0033 0.00210.0032 >0.0033 Doxorubicin 0.011 0.0039 0.014 0.028 0.022 0.016 0.0085Vincristine 0.0029 0.00049 0.00049 0.0024 0.0011 0.0060 0.00021Imatinib >0.7 >0.7 >0.7 >0.7 >0.7 >0.7 0.085

Example 3 Determination of IC₅₀ Values and Reduction in Dosage ofChemotherapeutic Agent in Mixtures of Steroid Saponins with Cisplatin,Docetaxel, Doxorubicin and Vincristine

The cell seeding and ELISA plate methodology of Example 1 were used fordetermining inhibition of two cancer cell lines. IC₅₀ values weredetermined for the steroid saponins dioscin, deltonin and prosapogeninA, and their mixtures with cisplatin, docetaxel, doxorubicin andvincristine, using LNCap and MCF7 cell lines.

The two-component mixtures were made up by mixing 50% of IC₅₀ values foreach component where the IC₅₀ values of the steroid saponins andchemotherapeutic agents are given in Table 6:

TABLE 6 IC₅₀ values used in assay (μM) LNCap MCF7 Dioscin 1 1 Deltonin 11 Prosapogenin A 2 2 Cisplatin 0.8 0.8 Docetaxel 0.00003 0.00003Doxorubicin 0.004 0.015 Vincristine 0.0005 0.0005

The mixtures were made up as illustrated in Table 7:

TABLE 7 Concentrations used for dioscin and cisplatin indioscin:cisplatin, 50:50 IC₅₀ mixture, against LNCap cell line ELISACell 1 2 3 4 5 6 7 8 Multiplying Factor 8 4 2 1 0.5 0.25 0.125 0 Dioscin(μM) 4 2 1 0.5 0.25 0.125 0.0625 0 Cisplatin (μM) 3.2 1.6 0.8 0.4 0.20.1 0.05 0

Thus, in ELISA cell number 4 (Multiplying Factor=1), the concentrationis 50% of each of the individual IC₅₀ values: 50% of 1 μM for dioscinand 50% of 0.8 μM for cisplatin.

Table 8 provides a further illustration of setting up ELISA cellconcentrations, where in ELISA cell number 4 (Multiplying Factor=1), theconcentration is 50% of each of the individual IC₅₀ values: 50% of 2 μMfor prosapogenin A and 50% of 0.015 μM for doxorubicin against MCF7:

TABLE 8 Concentrations used for dioscin and cisplatin in prosapogeninA:doxorubicin, 50:50 IC₅₀ mixture, against MCF7 cell line ELISA Cell 1 23 4 5 6 7 8 Multiplying Factor 8 4 2 1 0.5 0.25 0.125 0 Prosapogenin A 84 2 1 0.5 0.25 0.125 0 (μM) Doxorubicin (μM) 0.06 0.03 0.015 0.00750.00375 0.00188 0.00094 0

The inhibitory data from each mixture was used to determine an IC₅₀ ofIC₅₀ value, where the concentrations used were the Multiplying Factorvalues. In order to determine the actual IC₅₀ contribution of eachcomponent in the two-component mixture, the IC₅₀ of IC₅₀ value wasmultiplied by the IC₅₀ of the individual component. This can then beused to determine the reduction in IC₅₀ contribution (ie, reduction indose) of the chemotherapeutic agent due to the presence of the steroidsaponin in the mixture. A simpler way of determining the reduction indose is to use the following simple formula:

${{Reduction}\mspace{14mu} {in}\mspace{14mu} {dose}\mspace{14mu} (\%)} = \frac{( {{{IC}_{50}\mspace{14mu} {of}\mspace{14mu} {chemotherapeutic}\mspace{14mu} {agent}} - {0.5 \times ( {{IC}_{50}\mspace{14mu} {of}\mspace{14mu} {IC}_{50}} )}} ) \times 100}{{IC}_{50}\mspace{14mu} {of}\mspace{14mu} {chemotherapeutic}\mspace{14mu} {agent}}$

The following IC₅₀ of IC₅₀ values in Table 9 were determined for thesteroid saponins of this example:

TABLE 9 LNCap MCF7 IC₅₀ of IC₅₀ IC₅₀ of IC₅₀ Dioscin 0.56 1.1 Deltonin0.42 0.67 Prosapogenin A 0.89 1.1

The IC₅₀ of IC₅₀ values for the chemotherapeutic agents and for theirmixtures with the steroid saponins, plus the reduction in dose of eachchemotherapeutic agent determined when mixed with the steroid saponins,are given in Table 10:

TABLE 10 Cisplatin mixtures Cisplatin MCF7 Cisplatin LNCap Dose IC₅₀ ofDose IC₅₀ of IC₅₀ Reduction IC₅₀ Reduction Cisplatin 0.37 0.82Cisplatin + dioscin 0.54 27% 1.5 9% Cisplatin + deltonin 0.91 −23% 1.59% Cisplatin + 1.8 −143% 2.2 −34% prosapogenin A

There was no effective or consistent dose reduction in dosage ofcisplatin seen in mixtures of the steroid saponins with cisplatin.

Docetaxel mixtures LNCap Docetaxel MCF7 Docetaxel IC₅₀ of Dose IC₅₀ ofDose IC₅₀ Reduction IC₅₀ Reduction Docetaxel 3.7 8 Docetaxel + dioscin1.3 82% 3.2 80% Docetaxel + deltonin 0.97 87% 1.7 89% Docetaxel + 2.073% 2.0 88% prosapogenin A

There was a consistent reduction in dosage of docetaxel seen in mixturesof the steroid saponins with docetaxel.

Doxorubicin mixtures LNCap Doxorubicin MCF7 Doxorubicin IC₅₀ of DoseIC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Doxorubicin 1.4 1.6Doxorubicin + dioscin 0.89 68% 1.3 59% Doxorubicin + deltonin 1.1 61%2.0 38% Doxorubicin + 1.7 39% 2.5 22% prosapogenin A

There was a reduction in dosage of doxorubicin seen in mixtures of thesteroid saponins with doxorubicin, with dioscin and deltonin providing agreater reduction than prosapogenin A.

Vincristine mixtures LNCap Vincristine MCF7 Vincristine IC₅₀ of DoseIC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Vincristine 0.77 1.3Vincristine + dioscin 0.68 56% 1.8 31% Vincristine + deltonin 1.1 29%1.5 42% Vincristine + 1.8 −17% 2.1 19% prosapogenin A

There was a consistent reduction in dosage of doxorubicin seen inmixtures of dioscin and deltonin with vincristine, with prosapogenin Ashowing effectively zero reduction in vincristine dosage.

Example 4 Determination of IC₅₀ Values and Reduction in Dosage ofPaclitaxel in Mixtures of Steroid Saponins with Paclitaxel

The cell seeding and ELISA plate methodology of Example 1 were used fordetermining inhibition of two cancer cell lines. IC₅₀ values weredetermined for the steroid saponins dioscin, deltonin and prosapogeninA, and their mixtures with paclitaxel, using A549 and MCF₇ cell lines.

The two-component mixtures were made up in the same manner as in Example2, by mixing 50% of IC₅₀ values for each component where the IC₅₀ valuesof the steroid saponins and paclitaxel are given in Table 11:

TABLE 11 IC₅₀ values used in assay (μM) A549 MCF7 Dioscin 1 1 Deltonin 11 Prosapogenin A 2 2 Paclitaxel 0.003 0.001

Using the calculation methodology described in Example 2, the followingIC₅₀ of IC₅₀ values in Table 12 were determined for the steroid saponinsof this example:

TABLE 12 A549 MCF7 IC₅₀ of IC₅₀ IC₅₀ of IC₅₀ Dioscin 1.9 1.9 Deltonin0.70 1.2 Prosaspogenin A 0.99 1.0

The IC₅₀ of IC₅₀ values for paclitaxel and for its mixtures with thesteroid saponins, plus the reduction in dose of paclitaxel determinedwhen mixed with the steroid saponins, are given in Table 13:

TABLE 13 Paclitaxel mixtures Paclitaxel Paclitaxel LNCap Dose MCF7 DoseIC₅₀ of IC₅₀ Reduction IC₅₀ of IC₅₀ Reduction Paclitaxel 1.7 1.5Paclitaxel + dioscin 2.2 35% 1.9 37% Paclitaxel + deltonin 1.0 71% 1.550% Paclitaxel + 2.3 32% 2.3 23% Prosapogenin A

There was a reduction in dosage of paclitaxel seen in mixtures of thesteroid saponins with doxorubicin, with deltonin providing a greaterreduction than dioscin and prosapogenin A.

Example 5 Determination of IC₅₀ Values and Reduction in Dosage ofChemotherapeutic Agent in Mixtures of Steroid Saponins with Cisplatin,Docetaxel, Doxorubicin and Vincristine

The cell seeding and ELISA plate methodology of Example 1 were used fordetermining inhibition of four cancer cell lines. IC₅₀ values weredetermined for the steroid saponins dioscin, deltonin and prosapogeninA, and their mixtures with cisplatin, docetaxel, doxorubicin andvincristine, using PC3, DU145, A549 and HT29 cell lines.

The two-component mixtures were made up in the same manner as in Example2, by mixing 50% of IC₅₀ values for each component where the IC₅₀ valuesof the steroid saponins and chemotherapeutic agents are given in Table14:

TABLE 14 IC₅₀ values used in assay (μM) Dioscin 1 Deltonin 1Prosapogenin A 2 Cisplatin 0.8 Docetaxel 0.0002 Doxorubicin 0.015Vincristine 0.0025

Using the calculation methodology described in Example 2, the followingIC₅₀ of IC₅₀ values in Table 15 were determined for the steroid saponinsof this example:

TABLE 15 PC3 DU145 A549 HT29 IC₅₀ of IC₅₀ IC₅₀ of IC₅₀ IC₅₀ of IC₅₀ IC₅₀of IC₅₀ Dioscin 2.9 1.9 2.3 2.8 Deltonin 1.2 0.9 0.9 0.9 Prosapogenin1.5 1.5 1.5 1.5 A

The IC₅₀ of IC₅₀ values for the chemotherapeutic agents and for theirmixtures with the steroid saponins, plus the reduction in dose of eachchemotherapeutic agent determined when mixed with the steroid saponins,are given in Tables 16 to 19:

TABLE 16 Cisplatin mixtures PC3 Cisplatin DU145 Cisplatin IC₅₀ of DoseIC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Cisplatin 2.7 1.6 Cisplatin +dioscin 3.5 35% 2.9  9% Cisplatin + deltonin 1.7 69% 1.9 41% Cisplatin +prosapogenin A 2.0 63% 2.1 34% A549 Cisplatin HT29 Cisplatin IC₅₀ ofDose IC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Cisplatin 1.7 1.5Cisplatin + dioscin 3.0 12% 3.0  0% Cisplatin + deltonin 1.7 50% 1.8 40%Cisplatin + prosapogenin A 2.4 29% 2.8  7%

With cisplatin there was a reduction in dosage provided in all casesexcept for minimal to zero reduction for cisplatin+dioscin with DU145and with HT29, and minimal reduction for cisplatin+prosapogenin A withHT29.

TABLE 17 Docetaxel mixtures PC3 Docetaxel DU145 Docetaxel IC₅₀ of DoseIC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Docetaxel 2.9 3.1 Docetaxel +dioscin 3.9 33% 3.8 39% Docetaxel + deltonin 2.2 62% 1.9 69% Docetaxel +prosapogenin A 2.6 55% 2.4 61% A549 Docetaxel HT29 Docetaxel IC₅₀ ofDose IC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Docetaxel 3.9 3.3Docetaxel + dioscin 3.8 51% 3.6 45% Docetaxel + deltonin 1.7 78% 1.9 71%Docetaxel + prosapogenin A 3   62% 3   55%

With docetaxel there was a reduction in dosage provided by each of thesteroid saponin mixtures with each of the four cell lines as illustratedin Table 17.

TABLE 18 Doxorubicin mixtures PC3 Doxorubicin DU145 Doxorubicin IC₅₀ ofDose IC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Doxorubicin 5.5 3.1Doxorubicin + dioscin 5   55% 3.4 45% Doxorubicin + deltonin 2.2 80% 2.363% Doxorubicin + 2.9 74% 2.3 63% prosapogenin A A549 Doxorubicin HT29Doxorubicin IC₅₀ of Dose IC₅₀ of Dose IC₅₀ Reduction IC₅₀ ReductionDoxorubicin 3.2 4.7 Doxorubicin + dioscin 2.3 64% 3.5 63% Doxorubicin +deltonin 1.8 72% 2.2 77% Doxorubicin + 2.7 58% 3   68% prosapogenin A

With doxorubicin there was a reduction in dosage provided by each of thesteroid saponin mixtures with each of the four cell lines as shown inTable 18.

TABLE 19 Vincristine mixtures PC3 Vincristine DU145 Vincristine IC₅₀ ofDose IC₅₀ of Dose IC₅₀ Reduction IC₅₀ Reduction Vincristine 3.2 8  Vincristine + dioscin 2.3 64% 3.5 78% Vincristine + deltonin 1.3 80% 1.889% Vincristine + 2.7 58% 2.5 84% prosapogenin A A549 Vincristine HT29Vincristine IC₅₀ of Dose IC₅₀ of Dose IC₅₀ Reduction IC₅₀ ReductionVincristine 8   7.5 Vincristine + dioscin 3.1 81% 4.1 73% Vincristine +deltonin 1.7 89% 2.2 85% Vincristine + 3   81% 3   80% prosapogenin A

With vincristine there was a reduction in dosage provided by each of thesteroid saponin mixtures with each of the four cell lines as shown inTable 19.

Example 6 Determination of IC₅₀ Values and Reduction in Dosage ofImatinib in Mixtures of Steroid Saponins with Imatinib

The cell seeding and ELISA plate methodology of Example 1 were used fordetermining inhibition of the K562 cell line. IC₅₀ values weredetermined for the steroid saponins dioscin, deltonin and prosapogeninA, and their mixtures with imatinib, using the K562 cell line.

The two-component mixtures were made up in the same manner as in Example2, by mixing 50% of IC₅₀ values for each component where the IC₅₀ valuesof the steroid saponins and imatinib are given in Table 20:

TABLE 20 IC₅₀ values used in assay (μM) Dioscin 1 Deltonin 1Prosapogenin A 2 Imatinib 0.09

Using the calculation methodology described in Example 2, the followingIC₅₀ of IC₅₀ values were determined for the steroid saponins of thisexample and shown in Table 21:

TABLE 21 K562 IC₅₀ of IC₅₀ Dioscin 1.2 Deltonin 0.69 Prosapogenin A 1.2

The IC₅₀ of IC₅₀ values for imatinib and for its mixtures with thesteroid saponins, plus the reduction in dose of imatinib determined whenmixed with the steroid saponins, are given in Table 22:

TABLE 22 Imatinib mixtures K562 Imatinib IC₅₀ of IC₅₀ Dose ReductionImatinib 1.6 Imatinib + dioscin 1.4 56% Imatinib + deltonin 1.1 66%Imatinib + prosapogenin A 2 38%

With imatinib there was a reduction in dosage provided by each of thesteroid saponin mixtures with the K562 cell line.

Example 7 Determination of Degree of In Vivo Enhancement of AnticancerActivity of a Chemotherapeutic Drug when Co-Administered with a SteroidSaponin

5-Fluorouracil (5FU) is the principal chemotherapeutic drug used intreating colon cancer; it is most commonly co-administered with otherchemotherapeutic agents. This study compared the co-administration of5-fluoruracil and deltonin, with administration of mono-administrationof both 5-fluorourcil and deltonin.

72 female Balb/c nude female mice were microchipped, weighed andrandomised based on body weight into 8 groups with 9 mice per group.

The treatments, which were all formulated in NMP:PEG300:Water (1:9:10,v/v) and administered daily by intravenous injection for 5-fluorouraciland otherwise by intraperitoneal injection, were as given in thefollowing table (NMP═N-methylpyrrolidone):

TABLE 23 Treatment Dose Administration 5-Fluoruracil 37.5 mg/kg weeklyi.p. 5-Fluoruracil + deltonin 37.5/1.65 mg/kg weekly i.v./daily i.p.5-Fluoruracil + deltonin 37.5/3.3 mg/kg weekly i.v./daily i.p.5-Fluoruracil + deltonin 37.5/6.6 mg/kg weekly i.v./daily i.p. Deltonin1.65 mg/kg daily i.p. Deltonin 3.3 mg/kg daily i.p. Deltonin 6.6 mg/kgdaily i.p.

HT29 human prostate carcinoma cells were cultured in RPMI1640 cellculture medium, which was supplemented with 10% FBS andpenicillin-streptomycin (50 IU/mL final concentration). The cells wereharvested by trypsinisation, washed twice in HBSS and counted. The cellswere then resuspended in HBSS and adjusted to a final volume containing2×10⁷ cells/mL. For inoculation, the needle was introduced through theskin into the subcutaneous space just below the right shoulder, where100 μL of cells (2×10⁶) were discharged.

When the tumour volumes had reached an average of greater than 200 mm³,treatment was started (Day 0). Tumour volumes were measured 3-timesweekly, and determined according to the formula:

V (mm³)=length×diameter²×π/6

Treatment was continued for 17 days. The mean tumour volumes for eachdosage regime are given in Table 24 and are presented graphically inFIG. 1:

TABLE 24 Days Treatment 0 2 4 7 9 11 14 16 18 5-Fluorouracil (5FU) 236280 307 330 432 511 681 676 824 (37.5 mg/kg) (1 × weekly) 5FU/deltonin216 207 249 294 255 339 386 441 523 (37.5/1.65 mg/kg) (1 × weekly/daily)5FU/deltonin 235 212 229 278 264 315 381 451 598 (37.5/3.3 mg/kg) (1 ×weekly/daily) 5FU/deltonin 236 206 244 281 292 339 440 532 543 (37.5/6.6mg/kg) (1 × weekly/daily) Deltonin (1.65 mg/kg) 236 260 299 294 350 462594 644 776 (daily) Deltonin (3.3 mg/kg) 236 242 271 331 413 489 558 742842 (daily) Deltonin (6.6 mg/kg) 235 233 265 310 399 490 588 671 692(daily)

The tumour volumes with the combined treatments of deltonin with5-fluoruracil are less than the tumour volumes of any of themono-treatments, that is, 5-fluorouracil alone, or any of the 3 deltonintreatments alone.

Finally, it will be appreciated that various modifications andvariations of the methods and compositions of the invention describedherein will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are apparent tothose skilled in the art are intended to be within the scope of thepresent invention.

1. A method of inhibiting growth of a cancerous cell, the methodincluding the step of exposing the cancerous cell to an anti-cancertherapy and an effective amount of a steroid saponin.
 2. A methodaccording to claim 1, wherein the steroid saponin includes a saccharideattached to a single position of the sapogenin component of the steroidsaponin.
 3. A method according to claim 2, wherein the saccharide isattached to the C-3 position of the sapogenin.
 4. A method according toclaim 2, wherein the saccharide includes one or more monosaccharideunits selected from D-glucose (Glc), L-rhamnose (Rha), D-galactose(Gal), D-glucuronic acid (GlcA), D-xylose (Xyl), L-arabinose (Ara),D-fucose (Fuc), D-galacturonic acid (GalA).
 5. A method according toclaim 1, wherein the sapogenin component of the steroid saponin is basedon a sapogenin selected from the group consisting of a spirostanol,including diosgenin, yamogenin (neodiosgenin), yuccagenin,sarsasapogenin, tigogenin, smilagenin, hecogenin, gitogenin,convallamarogenin, neoruscogenin, and solagenin; a furostanol includingprotodiosgenin, pseudoprotodiosgenin, methyl protodiosgenin,protoyamogenin and methyl protoyamogenin.
 6. A method according to claim1, wherein the steroid saponin is a chacotrioside-steroid saponin or asolatrioside-steroid saponin.
 7. (canceled)
 8. A method according toclaim 6, wherein the chacotrioside-steroid saponin is selected from thegroup consisting of diosgenin linked through the C-3 position tochacotriose; diosgenin linked through the C-3 position to anotherchacotrioside; tigogenin linked through the C-3 position to achacotrioside; sarsasapogenin linked through the C-3 position to achacotrioside; smilagenin linked through the C-3 position to achacotrioside; yuccagenin linked through the C-3 position to achacotrioside; and yamogenin linked through the C-3 position to achacotrioside, and wherein the solatrioside-steroid saponin is selectedfrom the group consisting of gracillin; deltonin; diosgenin solatriose;diosgenin linked through the C-3 position to another solatrioside;tigogenin linked through the C-3 position to a solatrioside;sarsasapogenin linked through the C-3 position to a solatrioside;smilagenin linked through the C-3 position to a solatrioside; yuccageninlinked through the C-3 position to a solatrioside; and yamogenin linkedthrough the C-3 position to a solatrioside.
 9. (canceled)
 10. A methodaccording to claim 1, wherein the steroid saponin has the chemicalformula:

wherein R1, R2, R4, R6, R7, R11, R12, R14, R15 and R17 are independentlyH, OH, ═O, pharmacologically acceptable ester groups orpharmacologically acceptable ether groups; R5 is H when C-5,C-6 is asingle bond, and nothing when C-5,C-6 is a double bond; A is either Oconcurrently with B being CH₂, or B is O concurrently with A being CH₂;R27A is H concurrently with R27B being CH3, or R27A is CH3 concurrentlywith R27B being H; R3 comprises a glycosyl group linked through theoxygen atom to the steroidal sapogenin at C-3; or a pharmaceuticallyacceptable salt, or derivative thereof.
 11. A method according to claim1, wherein the steroid saponin has the chemical formula:

wherein R1, R2, R4, R6, R7, R11, R12, R14, R15 and R17 are independentlyH, OH, ═O, pharmacologically acceptable ester groups orpharmacologically acceptable ether groups; R5 is H when C-5,C-6 is asingle bond, and nothing when C-5,C-6 is a double bond; R22 is either ahydroxyl or an alkoxyl group when C-20, C-22 is a single bond, ornothing when C-20, C-22 is a double bond; R_(27A) is H concurrently withR_(27B) being CH₃, or R_(27A) is CH₃ concurrently with R_(27B) being H;R28 is H or a saccharide; or a pharmaceutically acceptable salt, orderivative thereof; R3 comprises a glycosyl group linked through theoxygen atom to the steroidal sapogenin at C-3; or a pharmaceuticallyacceptable salt, or derivative thereof.
 12. A method according to claim1, wherein the steroid saponin is selected from the group consisting ofdiosgenin linked through the C-3 position to a saccharide, tigogeninlinked through the C-3 position to a saccharide, sarsasapogenin linkedthrough the C-3 position to a saccharide, smilagenin linked through theC-3 position to a saccharide, yuccagenin linked through the C-3 positionto a saccharide, and yamogenin linked through the C-3 position to asaccharide. 13.-17. (canceled)
 18. A method according to claim 1,wherein the steroid saponin is selected from the group consisting ofdeltonin (diosgenin Rha2, [Glc4], Glc), dioscin (diosgenin Rha2, [Rha4],Glc), prosapogenin A (diosgenin Rha2, Glc) and asperin (diosgenin [Rha4, Rha 4], Rha 2, Glc).
 19. A method according to claim 1, wherein theanti-cancer therapy is exposure of the cell to an anti-cancer agent, oris radiotherapy.
 20. (canceled)
 21. A method according to claim 19,wherein the anti-cancer agent is an agent selected from one or more ofthe group consisting of alkylating agents, including BCNU (carmustine),bisulfan, CCNU (lomustine), chlorambucil, cisplatin, melphan, mitomycinC, and thio-tepa; antimitotic agents including taxol (paclitaxel),docetaxel, vinblastine sulphate, and vincristine sulphate; topoisomeraseinhibitors including doxorubicin, daunorubicin, m-AMSA (amsacrine),mitoxantrone, and VP-16 (etoposide); RNA/DNA antimetabolites including5-fluorouracil and methotrexate; DNA antimetabolites including Ara-C(cytarabine), hydroxyurea (hydroxycarbamide), and thioguanine(tioguanine); a cellular process targeting agent; imatinib mesylate;trastuzumab; and gefitinib. 22.-29. (canceled)
 30. A method of promotingthe activity of an anti-cancer therapy in a subject, the methodincluding exposing the subject to an effective amount of a steroidsaponin. 31.-43. (canceled)
 44. A method of promoting apoptosis of acancerous cell due to exposure of the cancerous cell to an anti-cancertherapy, the method including exposing the cancerous cell to aneffective amount of a steroid saponin. 45.-54. (canceled)
 55. A methodaccording to claim 1, wherein the method is used to inhibit formationand/or growth of a tumor in a subject.
 56. A method according to claim1, wherein the method is used to prevent and/or treat cancer in asubject.
 57. A method according to claim 30, wherein the method is usedto reduce the amount of the anti-cancer therapy provided to the subjectto prevent and/or treat a cancer in the subject.
 58. A method accordingto claim 30, wherein the method is used to prevent and/or treat a cancerin the subject, wherein the subject has an increased resistance to theanti-cancer therapy.
 59. A method according to claim 44, wherein themethod reduces resistance developing to the anti-cancer therapy in thecancerous cell.